Regulation of bacterial autolytic enzymes (cell wall hydrolases) is a highly sophisticated physiological task. Antibiotics such as penicillin induce bacteriolysis by interfering with the control of the endogenous autolytic enzymes, indicating the major chemotherapeutic relevance of autolysins. Although the binding of antibiotics to cell wall synthetic enzymes has been very well characterized, it is unknown how this event leads to deregulation of autolytic enzymes. It is this aspect of antibiotic activity, revealed as the tolerant phenotype, that is the focus of this proposal. Bacteria which stop growing in response to penicillin but fail to lyse and die are termed tolerant. This property, first described in pneumococcus, ensures bacterial survival and is the first step for most strains on the way to development of antibiotic resistance. During the first 5 years of this proposal, 5 genetic loci were identified which produced tolerance when mutated in pneumococcus. These are the first members identified in an autolytic cascade. Two of the loci defined a signal transduction apparatus triggering autolysis. This proposal seeks to build on these findings by characterizing in detail the mechanism of signal transduction which results in lysis. In particular, the structure and metabolism of the death peptide signal will be elucidated and a potential second peptide signal will be characterized. To identify more elements in the autolysis cascade, two approaches will be taken. Two additional tolerant mutants will be studied in detail. Second, elements in the VncR regulon affected by the VncR DNA binding protein will be sought. Finally, the significance of tolerance in the clinical setting will be defined by improving diagnostics for this trait and investigating the impact of tolerance on the course of infection in animal models. This will provide information important to the development of new potential antibacterial agents and perhaps suggest why bacteria in the clinical environment choose to regulate autolytic activity rather than dispense with suicidal autolysins in the face of antibiotic pressure.